Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks’ C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway

Outer membrane protein a conservation among Orientia tsutsugamushi isolates suggests its potential as a protective antigen and diagnostic target

Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium; Orientia tsutsugamushi; . A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA) are highly conserved among 51; O. tsutsugamushi; isolates. Molecular modeling revealed the predicted tertiary structure of; O. tsutsugamushi; OmpA to be very similar to that of the phylogenetically-related pathogen,; Anaplasma phagocytophilum; , including the location of a helix that contains residues functionally essential for; A. phagocytophilum; infection. PCR primers were developed that amplified; ompA; DNA from all; O. tsutsugamushi; strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of; O. tsutsugamushi ompA; DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among; O. tsutsugamushi; strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine

ORIENTIA TSUTSUGAMUSHI ANKYRIN-REPEAT PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM

Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3

Anaplasma phagocytophilum-Occupied Vacuole Interactions with the Host Cell Cytoskeleton

Anaplasma phagocytophilum is an obligate intracellular bacterial pathogen of humans and animals. The A. phagocytophium-occupied vacuole (ApV) is a critical host-pathogen interface. Here, we report that the intermediate filaments, keratin and vimentin, assemble on the ApV early and remain associated with the ApV throughout infection. Microtubules localize to the ApV to a lesser extent. Vimentin, keratin-8, and keratin-18 but not tubulin expression is upregulated in A. phagocytophilum infected cells. SUMO-2/3 but not SUMO-1 colocalizes with vimentin filaments that surround ApVs. PolySUMOylation of vimentin by SUMO-2/3 but not SUMO-1 decreases vimentin solubility. Consistent with this, more vimentin exists in an insoluble state in A. phagocytophilum infected cells than in uninfected cells. Knocking down the SUMO-conjugating enzyme, Ubc9, abrogates vimentin assembly at the ApV but has no effect on the bacterial load. Bacterial protein synthesis is dispensable for maintaining vimentin and SUMO-2/3 at the ApV. Withaferin A, which inhibits soluble vimentin, reduces vimentin recruitment to the ApV, optimal ApV formation, and the bacterial load when administered prior to infection but is ineffective once vimentin has assembled on the ApV. Thus, A. phagocytophilum modulates cytoskeletal component expression and co-opts polySUMOylated vimentin to aid construction of its vacuolar niche and promote optimal survival

Nous ne sommes pas venus en vain, quelqu'un se souviendra de nous : l'installation comme tentative de réhumanisation du monde

Mon sujet de recherche en création est né d'une question demeurée sans réponse ; pourquoi Auschwitz ? À la suite de mes investigations, j'ai réalisé que ce n'est pas tant l'atrocité liée exclusivement au camp de la mort que j'interrogeais et tentais de comprendre, mais la déshumanisation de l'individu. Par déshumanisation j'entends dépersonnalisation. J'ai donc essayé de comprendre quels sont les dispositifs sociaux qui causent ce durcissement face à l'individu. Comment en arrive-t-on à départir une vie qui mérite d'être protégée, pleurée et une autre qui ne le mérite pas ? J'ai tenté de répondre à ma problématique de recherche initiale : «Comment redonner l'humanité aux individus par le biais de l'installation ?» en adoptant une perspective artistique. Dès le début de ma recherche, je me suis trouvée devant une impasse : les ouvrages théoriques sur le devoir de mémoire renvoient systématiquement aux guerres, aux génocides, à la « Grande Histoire », à la « Grande mémoire », celles des livres, alors que ce qui m'intéresse c'est la « petite mémoire ». Cette petite mémoire qui fait l'importance et l'unicité de chacun, cette masse de petites choses prônant notre différence les uns des autres. En m'inscrivant dans une démarche heuristique, j'ai démontré par un travail expérientiel, la pertinence de me concentrer sur la « petite mémoire » (Boltanski, 2002) et l'importance de commémorer l'unicité de chacun. À force d'expérimentations, une hypothèse de création a émergé et elle s'est déployée sous la forme de l'installation. J'ai donc délaissé la peinture et le dessin pour l'installation, dans le but d'offrir au spectateur une expérience de l'art différente de celle à laquelle je le conviais avant, dont l'oeuvre est étroitement liée au lieu d'exposition. Bien que le monument soit associé à la longévité, aux matériaux durables tels le marbre, la pierre, j'ai privilégié Fimpermanence pour mon oeuvre finale, car contrairement à la croyance répandue, les monuments de granit ne sont-ils pas conçus justement pour oublier, pour régler l'affaire une fois pour toute, alors qu'en utilisant des matériaux éphémères, nous serons obligés de nous rappeler ; il faudra refaire la prière, encore et encore, afin que la mémoire se perpétue. Pour la dernière étape de mon projet, j'ai fait une analyse dérivée de la phénoménologie structurale (Mucchielli, 1983). Cette méthode stipule que notre essence est, de façon spontanée et inconsciente, imprégnée dans chacun des gestes ayant transformé de façon intentionnelle la matière. C'est ainsi que j'ai pu saisir et cibler les intentions profondes de la communauté avec laquelle j'ai travaillé. Enfin, les principaux résultats de cette recherche en création ont culminé par la réalisation d'une d'installation in situ, qui s'est tenue dans le jardin du couvent de la Congrégation des Soeurs du Très Saint-Sacrement; le lieu étant inhérent à l'oeuvre, il était essentiel de réaliser l'exposition à cet endroit précis

cPLA₂ regulates the <i>A</i>. <i>phagocytophilum</i>-induced NLRC4 inflammasome.

<p>BMDMs (1 x10⁶ cells) from wildtype (WT) or cPLA₂-deficient mice were infected with <i>A</i>. <i>phagocytophilum</i> (MOI 10/50) for 18 hours. (A) cPLA₂ enzymatic activity was measured. Release of (B) PGE₂, (C) PGD₂, (D) TBXA_2, (E) IL-1β, (F) IL-18 and (G) IL-6 were measured by ELISA in the cell culture supernatants. (H) SDS-PAGE immunoblot (IB) of caspase-1 (p20) in the supernatants. pro-IL-1β and pro-IL-18 were detected in cell lysates. Student’s t test. *<i>P</i> < 0.05. NS–not significant. (-) non-stimulated.</p

A. phagocytophilum infection induces eicosanoid biosynthesis.

<p>(A) Heat map of deep sequencing analysis showing the expression of eicosanoid metabolism genes in murine BMDMs (1.5×10⁷ cells) infected with <i>A</i>. <i>phagocytophilum</i> (MOI50) for 18 hours. (B) Schematics of eicosanoid metabolism in murine macrophages. Eicosanoid biosynthesis occurs after the release of arachidonic acid from cell membranes by phospholipase A2 (<i>e</i>.<i>g</i>., cPLA₂). Arachidonic acid is converted to thromboxane and prostaglandins by cyclooxygenases (COX1/2), whereas 12‐HETE (12‐hydroxyeicosatetraenoic acid), 15‐HETE, 5‐HETE and leukotrienes are synthesized by lipoxygenases (12/15‐LOX and 5-LOX). (C-I) 1.5×10⁷ wildtype (WT) BMDMs were stimulated with <i>A</i>. <i>phagocytophilum</i> (MOI25) overnight. Cells were scraped followed by sonication. Enzymatic activities of (C) cPLA₂, (D) COX1 and (E) COX2 were measured. Levels of (F) arachidonic acid (AA), (G) PGE₂, (H) PGD₂, and (I) TBXA₂ in the supernatants of WT BMDMs infected with <i>A</i>. <i>phagocytophilum</i> (MOI50) were detected. Student’s t test. *<i>P</i> < 0.05. (-) non-stimulated.</p

The PGE₂-EP3 axis regulates activation of the NLRC4 inflammasome upon <i>A</i>. <i>phagocytophilum</i> infection.

<p>(A-B) Wildtype (WT) BMDMs (1 x10⁶ cells) were infected with <i>A</i>. <i>phagocytophilum</i> (MOI50) for 18 hours or <i>Salmonella</i> (MOI25) for 1 hour. RNA and protein levels of the PGE₂-EP3 receptor was measured by qRT-PCR and western blot (IB). β-actin was also detected in cell lysates. (C-E) WT BMDMs (1 x10⁶ cells) was pre-treated with the EP3 antagonist (L-798106) for 30 minutes at indicated concentrations followed by <i>A</i>. <i>phagocytophilum</i> colonization (MOI50) for 18 hours. The levels of (C) IL-1β and (D) IL-6 were measured in cell culture supernatants by ELISA. (E) Caspase-1 autoproteolysis in cell culture supernatants. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates by SDS-PAGE immunoblots (IB). (F-H) WT and <i>Ep3</i>^-/- BMDMs (1 x10⁶ cells) were infected with <i>A</i>. <i>phagocytophilum</i> (MOI 10/50) for 18 hours. The levels of (F) IL-1β, (G) IL-18 and (H) IL-6 in culture supernatants were measured by ELISAs. (I) Caspase-1 autoproteolysis in cell culture supernatants. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates by SDS-PAGE immunoblots (IB). (J-M) Naïve or LPS-primed (50 ng/ml) WT and <i>Ep3</i>^-/- BMDMs (1 x10⁶ cells) were infected with <i>Salmonella</i> (MOI25) for 1 hour. The levels of (J) IL-1β, (K) IL-18 and (L) IL-6 in cultured supernatants were measured by ELISA. (M) Caspase-1 autoproteolysis in cell culture supernatants. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates by SDS-PAGE immunoblots (IB). Student’s t test and ANOVA-Tukey. <i>*P</i> < .05; NS, not significant. (-) non-stimulated.</p

Chemical inhibition of cPLA₂ affects the <i>A</i>. <i>phagocytophilum</i>-induced NLRC4 inflammasome.

<p>Wildtype (WT) BMDMs (1 x10⁶ cells) pre-treated for 30 minutes with pharmacological inhibitors of secreted PLA₂ (sPLA₂) (LY315920–10μM), cPLA₂ (AACOCF3–10μM), phospholipase C (PLC) (U73122- 10μM) and phospholipase D (PLD) (FIPI– 0.3μM) and infected with <i>A</i>. <i>phagocytophilum</i> (MOI50) for 18 hours. (A) PGE₂, (B) PGD₂, (C) TBXA_2, (D) IL-1β, (E) IL-18 and (F) IL-6 levels were measured by ELISA in the cell culture supernatants. (G) SDS-PAGE immunoblot (IB) of caspase-1 p20. pro-IL-1β and pro-IL-18 detected in cell lysates. One way ANOVA-Tukey. *<i>P</i>< 0.05. NS–not significant. (-), non-stimulated.</p

COX2 restricts <i>A</i>. <i>phagocytophilum</i> infection <i>in vivo</i>.

<p><i>A</i>. <i>phagocytophilum</i> infection of WT (n = 20) and COX2 (<i>ptgs2)</i>^-/- (n = 10) mice. Bacterial load in the (A) peripheral blood of infected mice at day 15. (B) IL-18, (C) PGE₂, (D) PGD₂ and (E) TBXA₂ release in the serum of infected animals. (F-G) Splenomegaly for COX2 (<i>ptgs2</i>^-/-) mice infected with <i>A</i>. <i>phagocytophilum</i>. (H) Splenic architecture depicting the red (RP) and the white (WP) pulp during <i>A</i>. <i>phagocytophilum</i> infection. One-way ANOVA-Tukey; Student t test; *<i>P</i> < 0.05. NS–not significant. (-) non-stimulated.</p